CN114195862B - Polypeptide and application thereof - Google Patents

Abstract

The invention discloses a polypeptide and application thereof, and the amino acid sequence of the polypeptide is shown as SEQ ID NO.1-SEQ ID NO. 4. The polypeptide of the invention utilizes phage display technology, phage in-vitro cell screening and in-vivo tumor screening to obtain the affinity peptide of the targeted osteosarcoma HOS cell, and the targeting and specific binding of the polypeptide to the osteosarcoma HOS are proved by in-vitro fluorescence microscope observation and in-vivo living experiments. The polypeptide has potential application prospect in targeted treatment of tumors of osteosarcoma.

Description

Polypeptide and application thereof

Field of the art

The invention belongs to the technical field of biology, relates to a polypeptide and application thereof, and in particular relates to a polypeptide targeting an osteosarcoma HOS cell line and application thereof.

(II) background art

Osteosarcoma is the most common primary malignancy in humans, with five-year survival rates of less than 70% for patients without metastasis and less than 30% for patients with metastasis. In the last three decades, the treatment mode of osteosarcoma has not been significantly advanced, and the main treatment modes existing in clinic are only surgical excision and chemotherapy, so that the long-term survival rate of osteosarcoma patients has not been significantly changed in the last three decades.

Targeted therapy of osteosarcoma is still under investigation, and current research is mostly focused on germ line and somatic genetics of osteosarcoma, trying to find therapeutic targets by comparing genetic changes of osteosarcoma. But osteosarcoma targeted therapy based on genomic examination is costly and still in the exploratory stage. In addition, osteosarcomas have high levels of somatic structural variation and copy number changes, resulting in osteosarcomas with high heterogeneity and few repeated target mutations. Therefore, there is an urgent need to develop a new osteosarcoma targeted therapeutic strategy.

The phage display technology can be used for searching polypeptides of targeted cells, and has strong application value in the aspect of identifying affinity polypeptides of tumor cells. Several studies currently indicate that short oligopeptides derived from phage display can be used for targeted therapy of cancer as well as for accurate diagnosis in combination with drugs, fluorochromes, even nanoparticles.

In view of the advantages of targeting polypeptides for targeted therapy, screening a polypeptide capable of targeting osteosarcoma cells is of great importance in targeted therapy of osteosarcoma.

(III) summary of the invention

The invention aims to provide a polypeptide and application thereof, and the polypeptide has wide application prospect in targeted treatment of osteosarcoma by utilizing phage display technology and obtaining the affinity peptide of a targeted human osteosarcoma HOS cell line through in vitro cell screening and in vivo tumor screening.

The technical scheme adopted by the invention is as follows:

the invention provides a polypeptide, in particular a polypeptide targeting an osteosarcoma HOS cell line, wherein the amino acid sequence of the polypeptide is any one or a combination of at least two of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 from the N end to the C end, and preferably the amino acid sequence shown as SEQ ID NO. 1;

SEQ ID NO.1：TPPRVPLLTFGS；

SEQ ID NO.2：YPAAYHTLTREP；

SEQ ID NO.3：DSPTSLVLRARS；

SEQ ID NO.4：STLLADVRIPGS。

according to the invention, through constructing a phage library, a polypeptide sequence capable of specifically binding to a human osteosarcoma HOS cell line is screened from a random phage polypeptide library. And combining the sequences with HOS cells, screening out four amino acid sequences specifically combined with the HOS cells, wherein the amino acid sequence shown in SEQ ID NO.1 has the best effect of combining with the HOS cells.

The invention also provides a DNA fragment containing the polypeptide nucleotide sequence, a recombinant vector containing at least one copy of the DNA fragment, and a recombinant cell containing the recombinant vector, and recombinant genetically engineered bacteria prepared by transforming host bacteria with the recombinant cell; the recombinant vector takes M13KE phage as a basic vector, and the recombinant genetically engineered bacterium host bacterium is preferably escherichia coli ER2738.

The invention also provides a pharmaceutical composition comprising the DNA fragment, the recombinant vector or the recombinant cell, and a pharmaceutically acceptable carrier.

The invention provides an application of the polypeptide in preparing tumor localization diagnostic reagent, antitumor drug or tumor cell targeting preparation.

The polypeptide is usually modified when being connected with a drug, and preferably, gly-Gly-Ser-Cys connector is added at the C end of the polypeptide amino acid sequence.

The tumor is human osteosarcoma, or lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer, etc. which can express the receptor combined with the polypeptide.

Compared with the prior art, the invention has the beneficial effects that:

(1) According to the invention, the polypeptide sequence capable of being specifically combined with human osteosarcoma HOS cells is screened from a random phage polypeptide library, so that the best combination effect of the amino acid sequence shown in SEQ ID NO.1 and the HOS cells is determined;

(2) The polypeptide of the invention combines with drugs, fluorescent dyes, carriers and the like to play roles of tumor diagnosis, tumor resistance and the like.

(3) The polypeptide of the invention has wide application prospect in the aspects of potential high expression of osteosarcoma, lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer and the like and tumor treatment of the receptor combined with the polypeptide.

(IV) description of the drawings

FIG. 1, in vivo imaging, shows the aggregation of polypeptides in tumors; a is a photograph of an in vivo image, wherein Saline is injected with physiological Saline, CY5.5Only is injected with 200 mu M physiological Saline solution of Cy5.5-maleimide, CY5.5-Peptide C is injected with physiological Saline solution of polypeptide conjugate of sequence 2, CY5.5-Peptide S is injected with physiological Saline solution of polypeptide conjugate of random sequence, and CY5.5-OTP is injected with physiological Saline solution of polypeptide conjugate of sequence 1; b is a bar graph of fluorescence intensity for each group of mice.

FIG. 2, in vitro fluorescence microscopy, demonstrates the affinity of the polypeptide shown in SEQ ID NO.1 for osteosarcoma HOS cells; a is a fluorescence micrograph, wherein Cy5.5-OTP represents a polypeptide conjugate of sequence 1, cy5.5-Peptide C represents a polypeptide conjugate of sequence 2, cy5.5-Peptide S represents a polypeptide conjugate of random sequence Peptide S, and Cy5.5only represents Cy5.5-maleimide; DAPI represents DAPI staining, FITC-phalloidin represents FITC-phalloidin staining, cy5.5-peptide peptides represent different cy 5.5-polypeptide conjugates, MERGE represents a combined image of the 3 staining channels described above; b is a bar graph of fluorescence intensity for each group.

FIG. 3, in vitro fluorescence microscopy, demonstrates the specificity of binding of the polypeptide shown in SEQ ID NO.1 to osteosarcoma HOS cells; a is a fluorescence micrograph, wherein HOS represents HOS cells, HUVECs represents control cells, HUVECs represents DAPI staining, cy5.5-OTP represents a Cy5.5-polypeptide conjugate of sequence 1, and MERGE represents a combined image of the 2 staining channels; b is a bar graph of fluorescence intensity for each group.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

the experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The composition of the reagent used in the invention is as follows:

LB liquid medium: 10g of NaCl,10g of tryptone, 5g of yeast extract, dissolved in 1L of ddH ₂ O. Steam sterilizing at 121deg.C for 20min.

LB agar medium: 10g of NaCl,10g of tryptone, 5g of yeast extract, 12g of Agar (Agar), dissolved in 1L of ddH ₂ O. Steam sterilizing at 121deg.C for 20min.

Bacterial culture plates: firstly, preparing isopropyl-beta-D-thiogalactoside (IPTG) and galactoside (X-gal) into 20mg/ml solutions by Dimethylformamide (DMF), and preserving the solutions at-20 ℃ in a dark place; then, 100. Mu.L of the above IPTG solution and X-gal solution were added to 100ml of LB agar medium, respectively, to prepare bacterial culture plates.

Washing buffer: serum-free DMEM cell culture medium of 0.5mg/ml bovine serum albumin and 0.1% (v/v) Tween 20.

Elution buffer: 0.1M glycine, 1mg/mL bovine serum albumin, dissolved in ddH ₂ O, the pH value is adjusted to 2.2 by using concentrated hydrochloric acid with the mass concentration of 36 percent.

Cell lysate: 0.02mg/ml sodium deoxycholate, 10mM Tris-HCl,2mM EDTA, dissolved in ddH ₂ O。

Tris-HCl buffer (Tris-hydroxymethyl aminomethane-HCl): 121.1g Tris,1L ddH ₂ And (3) regulating the pH value to 9.1,4 ℃ by using concentrated hydrochloric acid with the mass concentration of 36% and storing.

PEG/NaCl solution: 100g polyethylene glycol (PEG), 116.9g NaCl and 475mL ddH ₂ O。

NAP buffer: 80mM NaCl,50mM NH ₄ H ₂ PO ₄ The solvent is ddH ₂ O, 36% mass concentration concentrated hydrochloric acid to pH 7.0.

Tris Buffer Salt (TBS): 2.42g/L Tris (Tris (hydroxymethyl aminomethane), 29.22g/L NaCl,1L ddH) ₂ O, 36% mass concentration concentrated hydrochloric acid to pH 7.5.

M13KE phage library: purchased from Bio-lab biotechnology limited.

The room temperature is 25-30 ℃. Human osteosarcoma HOS cells were purchased from the chinese sciences Shanghai cell bank.

The polypeptides referred to in the examples below were synthesized and purified by Jiangsu Nanjing Jinsri biotechnology Co., ltd, and the sequencing was performed by Hubei Wuhan Baizhi biotechnology Co., ltd.

Example 1: the human osteosarcoma HOS cell line targeting peptide is obtained by in vitro cell screening and in vivo tumor screening by adopting phage display technology.

1. In vitro cell screening of human osteosarcoma HOS targeted phage

(1) Preparation of E.coli solution

(1) Thawing ER2738 E.coli bacterial solution at room temperature (about 10 ¹⁰ mu.L of the bacterial liquid was added to 20mL of LB medium, and the mixture was placed at 220rpm and shaking-cultured at 37℃for 4 hours until OD=500。

(2) 500g, centrifuging at 4 ℃ for 6min, and precipitating escherichia coli; precipitation bacterial particles were gently resuspended with 10mL of 80mM aqueous NaCl solution;

(3) shaking the resuspended solution at 50rpm of a shaking table at 37 ℃ for 20min, transferring the solution into a 50mL centrifuge tube, and centrifuging at 4 ℃ for 6min with 500 g; the pellet was resuspended in 1mL of pre-chilled NAP buffer at 4℃to give E.coli solution (OD=500). And then stored in a refrigerator at 4 ℃ to wait for use.

(2) In vitro screening of HOS cell targeting peptides of human osteosarcoma

(1) Human osteosarcoma HOS cells (50X 10) ⁶ And then inoculating into 10cm cell culture flask containing 10mL DMEM culture solution containing 10% fetal bovine serum by volume concentration, placing into cell culture box at 37deg.C and 5% CO ₂ After incubation to 90% cell density, an appropriate amount of M13KE phage library (about 1X 10) was added to the broth ¹⁰ And then incubated at 37℃for 1h.

(2) The culture broth was discarded, and 4mL of a pre-chilled washing buffer at 4℃was added to the flask and washed for 1 minute.

(3) The wash buffer was discarded, 5ml of PBS was added, and the cells were scraped with a cell scraper. The scraped cells were transferred to a 15mL centrifuge tube, centrifuged at 200g at 4℃for 10min, and the supernatant was discarded. 200 mu L of cell lysate is added, and the mixture is uniformly mixed to obtain the HOS cell affinity phage solution for human osteosarcoma.

(4) And (3) adding 1mL of the escherichia coli bacterial solution prepared in the step (1) into 1mL of the human osteosarcoma cell affinity phage solution, uniformly mixing, and culturing for 15min at room temperature to obtain phage-infected bacteria. Transferring into a 250mL conical flask, adding 20mL of LB culture solution, and placing in a shaking table, culturing at 220rpm and 37 ℃ overnight to obtain amplified phage solution.

(3) Phage purification

(1) Centrifuging 20mL of amplified phage solution at 4 ℃ and 3000g for 10min; the clear supernatant was transferred to another centrifuge bottle, 6mL of PEG/NaCl solution was added, and the mixture was allowed to stand at 4℃overnight, designated as solution A.

(2) Solution A was centrifuged at 5000g for 10min, the phage was precipitated, the supernatant was discarded, resuspended in 1.5mL TBS solution, 150. Mu.L PEG/NaCl solution was added and left overnight at 4℃and designated as solution B.

(3) Centrifuging the solution B at 5000g and 4 ℃ for 10min; the supernatant was discarded, and then 1mL of TBS solution was added to dissolve phage particles, thereby obtaining a purified phage solution.

2. In vivo screening of HOS targeting peptides for human osteosarcoma

(1) Nude mice HOS cell nodulation molding: will be about 10 ⁶ The HOS cells were injected subcutaneously into the left armpit of nude mice.

(2) To a tumor size of about 300cm ³ When the tail vein was injected with 100. Mu.L of the phage solution purified in the above steps 1- (3) - (3).

(3) After 24 hours, tumor tissue was isolated.

(4) The tumor tissue was thoroughly homogenized by shearing, grinding, adding 10ml of PBS, and homogenizing with a homogenizer.

(5) 1000g,4 ℃, for 10min, centrifuging the homogenate, taking the supernatant and discarding the tissue precipitate.

(6) And (3) adding 1mL of the escherichia coli solution prepared in the step (1) in advance into the supernatant, and incubating for 15min at room temperature to obtain a phage infected bacterial solution.

(7) In a 250mL conical flask, the phage-infected bacterial solution was transferred into LB medium, and cultured overnight on a shaker at 37℃and 220rpm to obtain amplified phage solution.

(8) Phage purification was as in step 1- (3).

3. Polypeptide sequence detection

After 4 rounds of the in vitro screening process, the in-vivo screening process of 1 round is carried out. Each round of purified phage solution was inoculated into a bacterial culture plate, incubated overnight at 37 ℃ and then blue monoclonal colonies were selected for sequencing. The sequencing results are shown in table 1,4 polypeptide sequences of the affinity osteosarcoma cells are obtained by screening, wherein the polypeptide sequence 1: TPPRVPLLTFGS (SEQ ID NO. 1), polypeptide sequence 2: YPAAYHTLTREP (SEQ ID NO. 2), polypeptide sequence 3: DSPTSLVLRARS (SEQ ID NO. 3), polypeptide sequence 4: STLLADVRIPGS (SEQ ID NO. 4).

TABLE 1 frequency of occurrence of polypeptide sequences

Example 2, human osteosarcoma HOS cell line targeting and specificity verification of polypeptide sequences.

1. In vivo validation of polypeptide targeting to osteosarcoma tissue

(1) During the synthesis of the polypeptides (sequence 1, sequence 2) selected in example 1, gly-Gly-Gly-Ser-Cys linker was added to the C-terminus of each polypeptide.

(2) After polypeptide synthesis, 1mL of 10mM Cy5.5-maleimide (dissolved in DMSO) was added to 1mL of 10mM polypeptide aqueous solution and reacted at room temperature for 3h. Cys of the polypeptide is conjugated to maleimide of the Cy5.5 dye to form a Cy5.5-polypeptide conjugate, wherein the polypeptide conjugate formed by sequence 1 is denoted as Cy5.5-OTP and the polypeptide conjugate formed by sequence 2 is denoted as Cy5.5-PeptideC. Meanwhile, a random sequence Peptide S (LTPSTRLPGFPV, SEQ ID NO. 5) is synthesized as a control, and is marked as Cy5.5-Peptide S after being combined with Cy5.5-maleimide.

(3) 200. Mu.M of physiological Saline solution (Cy5.5-OTP, cy5.5-Peptic S) of different Cy5.5-polypeptide conjugate, 200. Mu.M of physiological Saline solution (Cy5.5 only) of Cy5.5-maleimide and 100. Mu.L of physiological Saline (Saline) were respectively injected into HOS tumor-forming mice via tail vein, and the aggregation of the polypeptide in tumor was observed by a small animal living imaging system (PerkinElmer IVIS Spectrum CT) under the anesthesia state of the mice after 24 hours (FIG. 1A). Wherein the stronger the fluorescent signal at the tumor, the stronger the targeting ability of the polypeptide (B in FIG. 1).

The experimental results are shown in fig. 1: polypeptide sequence 1 (TPPRVPLLTFGS) and polypeptide sequence 2 (YPAAYHTLTREP) have targeting effect on osteosarcoma, wherein the targeting effect of polypeptide sequence 1 (TPPRVPLLTFGS) is optimal, and the fluorescence signal intensity of the polypeptide sequence 1 in tumor is obviously higher than that of other polypeptides, which indicates that the polypeptide sequence 1 has good targeting capability on tumor.

2. Verification of affinity specificity of polypeptide for osteosarcoma HOS cells

Since the amino acid sequence shown in SEQ ID No.1 has the best effect, we have subsequently verified the targeting and specificity of the amino acid sequence shown in SEQ ID No.1 to HOS cells in vitro experiments. The Cy5.5-polypeptide conjugates of sequences 1 and 2 (denoted as Cy5.5-OTP and Cy5.5-Peptide C) and the polypeptide conjugate of random sequence Peptide S (denoted as Cy5.5-Peptide S) were prepared as controls according to the method of step 1, while 1mM of a DMSO solution of Cy5.5-maleimide was used as a blank control, denoted as Cy5.5 Only.

(1) HOS cells were grown at 1.0X10 per well ⁶ The individual cells were inoculated into 24-well plates (1 ml of DMEM high-sugar medium containing 10% fetal calf serum by volume concentration), 37℃and 5% CO ₂ Culturing to 50% cell density.

(2) Blocking with 10% BSA was performed at 37℃for 60 min. The blocking solution was discarded and washed 3 times with PBS buffer.

(3) Cy5.5-OTP, cy5.5-Peptide S and Cy5.5-Peptide C were added to the cells at a final concentration of 10. Mu.M, respectively, and Cy5.5Only was incubated with HOS cells for 30 minutes. After washing 3 times with PBS buffer, FITC-phalloidin was stained for 1h, DAPI was stained for 5min, and the results of the experiments were observed under a fluorescence microscope at 37℃and shown in FIG. 2: the signal of OTP in HOS cells was significantly higher than that of control polypeptides, indicating that OTP has significant affinity for HOS cells.

3. Verification of binding specificity of OTP to osteosarcoma HOS cells

HOS cells and HUVECs (from Shanghai cell Bank of the national academy of sciences) were inoculated into 24-well plates (1.0X10 per well) respectively ⁶ Individual cells, 1ml DMEM high sugar medium containing 10% fetal bovine serum by volume concentration), were blocked with 10% bsa at 37 ℃ for 60 minutes, and then stained with cy5.5-OTP (10 μm) prepared in step 1 for 30 minutes, respectively. After three washes, DAPI was stained for 5min and observed under a fluorescence microscope at 37 ℃. The experimental results are shown in fig. 3: OTP aggregates significantly more in HOS cells than HUVECs, indicating that OTP can specifically bind to HOS cells.

In conclusion, the polypeptide and the product thereof can be combined with human osteosarcoma HOS cells in a targeting way, so that the polypeptide has wide application prospects in the aspects of targeted therapy and diagnosis of osteosarcoma, and can be widely applied to the aspects of targeted therapy of tumors with potential high expression of binding receptors of the polypeptide, such as lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer.

The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.

Sequence listing

<110> university of Zhejiang

<120> a polypeptide and use thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Unknown (Unknown)

<400> 1

Thr Pro Pro Arg Val Pro Leu Leu Thr Phe Gly Ser

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Unknown (Unknown)

<400> 2

Tyr Pro Ala Ala Tyr His Thr Leu Thr Arg Glu Pro

1 5 10

<210> 3

<211> 12

<212> PRT

<213> Unknown (Unknown)

<400> 3

Asp Ser Pro Thr Ser Leu Val Leu Arg Ala Arg Ser

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Unknown (Unknown)

<400> 4

Ser Thr Leu Leu Ala Asp Val Arg Ile Pro Gly Ser

1 5 10

Claims (9)

1. A polypeptide, which is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1; SEQ ID NO.1: TPPRVPLLTFGS.

2. A DNA fragment comprising a nucleotide sequence encoding the polypeptide of claim 1.

3. A recombinant vector comprising at least one copy of the DNA fragment of claim 2.

4. A recombinant cell comprising the recombinant vector of claim 3.

5. A pharmaceutical composition for the treatment of a tumor, comprising the polypeptide of claim 1, the DNA fragment of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4.

6. The pharmaceutical composition for treating tumors of claim 5, which further comprises a pharmaceutically acceptable carrier.

7. Use of the polypeptide of claim 1 for the preparation of a tumor localization diagnostic reagent, wherein the tumor is osteosarcoma.

8. Use of the polypeptide of claim 1 for the preparation of an anti-tumor medicament, wherein the tumor is osteosarcoma.

9. Use of the polypeptide of claim 1 for the preparation of a tumor cell targeting agent, wherein the tumor is osteosarcoma.